RESUMO
Cycloleucine was used to inhibit the formation of internal N6-methyladenosine residues in the messenger ribonucleic acid transcripts from cultured methotrexate resistant mouse sarcoma cells. Cells cultured in cycloleucine produced transcripts deficient in N6-methyladenosine residues and the 2'-O-methylated nucleosides of the cap structure; however, the formation of the 7-methylguanine nucleoside of the cap was not effected. Cytoplasmic polyadenylated transcripts were isolated from cells which had been pretreated with media containing cycloleucine and translated in an in vitro translation assay. The levels of translated dihydrofolate reductase were then analyzed by polyacrylamide gel electrophoresis. The amount of dihydrofolate reductase protein produced from the transcripts of the cycloleucine treated cells was 20% less than untreated transcripts. Ribonuclease protection assays demonstrated little difference in the cytoplasmic levels of dihydrofolate reductase transcripts between treated and untreated cells suggesting that the decrease in translation efficiency was not caused solely by an alteration in the processing or cytoplasmic transport of the transcripts. Translation of in vitro transcribed transcripts showed the presence of 2'-O-methylated nucleosides in the cap structure had a negative effect on translation efficiency, demonstrating that the results observed from cycloleucine treatment could not be due to the inhibition of 2'-O-methylation in the cap. These experiments therefore suggest that an inhibition of N6-methyladenosine residues in dihydrofolate reductase transcripts significantly alters their rate of translation.
Assuntos
Adenina/análogos & derivados , Biossíntese de Proteínas/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/genética , Adenina/antagonistas & inibidores , Animais , Cicloleucina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Proteínas de Neoplasias/análise , Nucleosídeos/análise , Capuzes de RNA/genética , Processamento Pós-Transcricional do RNA/genética , Ribonucleases , Tetra-Hidrofolato Desidrogenase/metabolismo , Células Tumorais CultivadasRESUMO
The effect of amino acid substitutions introduced to the third alpha-helix in bovine GH (bGH) was investigated. A GH analog (bGH-M8), in which three amino acids were substituted to form an idealized amphiphilic alpha-helix, possessed the same specific binding affinity as wild-type bGH to cell membranes prepared from 3T3-F442A cells or rat adipocytes. However, bGH-M8 failed to stimulate preadipocyte differentiation, as measured by the level of glycerol-3-phosphate dehydrogenase activity. An equimolar concentration of bGH-M8 was inhibitory for this adipogenic effect caused by bGH at a concentration of 30 pM. bGH-M8 also failed to induce an insulin-like response and reduced lipolytic potency in rat primary adipocytes. A 10-fold excess of bGH-M8 abolished the effect of wild-type bGH in the insulin-like and lipolytic assays. Thus, bGH-M8 inhibited these actions of wild-type bGH and, therefore, appears to be a competitive antagonist. These results suggest that a major biologically active domain resides in the third alpha-helix of bGH, which is independent of amino acids important in the initial interaction of GH with its receptor.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Hormônio do Crescimento/antagonistas & inibidores , Insulina/farmacologia , Lipólise/efeitos dos fármacos , Células 3T3 , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Hormônio do Crescimento/análogos & derivados , Masculino , Camundongos , RatosRESUMO
We have previously shown that a bovine (b) GH antagonist, bGH-M8, which possesses three amino acid substitutions in its third alpha-helix, inhibits mouse 3T3-F442A preadipocyte differentiation. In the current studies, we used the bGH and human (h) GH analogs with single amino acid substitution, bGH-G119R and hGH-G120R, for determining their biological activity using the preadipocyte differentiation assay. Short-term and long-term GH-inducible events were studied during adipose differentiation, including late marker gene expression (adipocyte protein 2), immediate early gene induction (c-fos), and tyrosine phosphorylation of intracellular proteins. The results demonstrated that these GH analogs not only failed to induce these three events, but also antagonized GH induction of c-fos expression and phosphorylation of proteins of apparent molecular mass of 95 kDa. Our present study agrees with the notion that GH must bind to the GH receptor via site one and with a second GH receptor molecule (or with some yet unidentified 'second target') through GH binding site two. This interaction is important for subsequent GH-dependent biological events.
Assuntos
Adipócitos/citologia , Hormônio do Crescimento/análogos & derivados , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Proteínas Recombinantes , Adipócitos/efeitos dos fármacos , Animais , Northern Blotting , Proteínas de Transporte/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/genética , Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Líquido Intracelular/metabolismo , Camundongos , Proteína P2 de Mielina/genética , Fosforilação , Proteínas Proto-Oncogênicas c-fos/genética , Tirosina/metabolismoAssuntos
Patógenos Transmitidos pelo Sangue , Odontólogos , Controle de Infecções/métodos , Transmissão de Doença Infecciosa do Paciente para o Profissional/prevenção & controle , Precauções Universais , Dispositivos de Proteção dos Olhos , Luvas Cirúrgicas , Infecções por HIV/transmissão , Hepatite B/prevenção & controle , Hepatite B/transmissão , Humanos , Transmissão de Doença Infecciosa do Profissional para o Paciente/prevenção & controle , Máscaras , Exposição Ocupacional , Roupa de Proteção , Sociedades Odontológicas , Suíça , VacinaçãoAssuntos
Clínicas Odontológicas/legislação & jurisprudência , Administração da Prática Odontológica/legislação & jurisprudência , Clínicas Odontológicas/organização & administração , Arquitetura de Instituições de Saúde/legislação & jurisprudência , Humanos , Higiene , Controle de Infecções/legislação & jurisprudência , Controle de Infecções/organização & administração , Administração da Prática Odontológica/organização & administração , Sociedades Odontológicas , SuíçaAssuntos
Assistência Odontológica/métodos , Esterilização/métodos , Terminologia como Assunto , Assistência Odontológica/instrumentação , Assistência Odontológica/normas , Europa (Continente) , Controle de Qualidade , Sociedades Odontológicas , Esterilização/instrumentação , Esterilização/normas , SuíçaRESUMO
The role of the cnxH+ gene specified polypeptide in the formation and function of the NADPH-nitrate reductase in Aspergillus nidulans was examined with the use of two complementing mutant strains which were grown as forced heterocaryons in the presence of nitrate. The niaD-421 structural gene mutant and the cnxH-318 co-factor gene mutant produce two components of the NADPH-cytochrome c reductase co-activity which can be distinguished by their enzymatic and physical behavior. This combination enabled us to isolate the de novo synthesis of niaD+ gene specified protomers from the constitutively formed co-factor at two stages of development. The proportion of induced and constitutively formed protomers in the isolated holoenzyme was measured after pulsing with [3H]-histidine or [14C]-histidine prior to induction with nitrate. The newly formed nitrate reductase was resolved by agarose gel electrofocusing and activity staining. In vivo assembly of a 7.8s enzyme in the heterocaryotic mycelium of the above strains is apparently achieved by the convener action of the cnxH+ gene directed polypeptide from the niaD- strain on the niaD+ gene directed protomers of the cnxH- partner. This occurs with or without Mo as a co-factor.
Assuntos
Aspergillus nidulans/enzimologia , Genes Fúngicos , Nitrato Redutases/biossíntese , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Histidina/metabolismo , Focalização Isoelétrica , Molibdênio/metabolismo , Mutação , NADPH-Ferri-Hemoproteína Redutase/biossíntese , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Nitrato Redutase (NAD(P)H) , Nitrato Redutases/metabolismoRESUMO
An analog of bovine growth hormone (bGH-M8: [Leu117, Arg119, Asp122]-bGH) with an idealized amphiphilic third alpha-helix has been proposed to be a functional antagonist of GH. In accordance with this proposition, bGH-M8 profoundly inhibited bGH-stimulated lipolysis by chicken adipose tissue in vitro. bGH-M8 alone was a weak agonist in the lipolytic assay (1.9% the potency of bGH). The present evidence indicates that bGH-M8 is a competitive antagonist of the lipolytic action of GH based upon the following results: (i) increasing concentrations of bGH-M8 (antagonist) produce progressively greater inhibition of GH-stimulated lipolysis; (ii) increasing concentrations of bGH (agonist) are capable of overcoming this antagonism; and (iii) Schild plot analysis (slope = -0.94) suggests a receptor antagonist with an equilibrium dissociation constant (KB) of 4.54 nM. In contrast to the antagonistic effects of bGH-M8 on bGH-stimulated lipolysis, bGH-M8 retained full insulin-like ("antilipolytic") activity (i.e., inhibition of glucagon-induced lipolysis). bGH-M8 and bGH were similarly potent in eliciting antilipolytic effects in vitro. Moreover, the antilipolytic effects of bGH-M8 and bGH were additive. Therefore, the third alpha-helix (particularly residues 117, 119, and 122) of bGH contains major structural determinants for the lipolytic effects of GH. The ability of bGH-M8 to act as an antagonist for at least one action of GH (lipolysis) while being a full agonist for another (antilipolysis) suggests that different domains of GH are responsible for its various biologic activities, possibly involving different binding sites and/or signal transduction mechanisms.
Assuntos
Hormônio do Crescimento/análogos & derivados , Hormônio do Crescimento/antagonistas & inibidores , Insulina/farmacologia , Lipólise/efeitos dos fármacos , Animais , Ligação Competitiva , Galinhas , Hormônio do Crescimento/química , Hormônio do Crescimento/farmacologia , Masculino , Camundongos , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
A synthetic oligonucleotide, 5'-d(CTAGT-CTAGACTAG)-3' which encodes translational termination codons in three reading frames, was inserted into either exon IV (pbGH-4A) or V (pbGH-5A) of the bovine growth hormone gene. The resultant plasmids, under the transcriptional regulation of the mouse metallothionein 1 promoter, were introduced into cultured mouse L-cells or rat GH3 cells. Compared to wild type bGH RNA, bGH-specific RNA transiently expressed from pBGH-5A or pBGH-4A DNA in mouse L-cells was similar or slightly smaller in size, respectively. Unexpectedly, bGH-4A RNA lacked exon IV sequences. Immunofluorescence and immunoprecipitation analyses revealed that wild type bGH was localized to the Golgi apparatus, while truncated hormones were confined to the cytoplasmic compartment of transfected cells. In addition, truncated hormones were shown to be secretion-defective albeit the bGH signal peptide was efficiently and precisely processed. Thus, structural alterations in the bGH gene can dramatically affect bGH precursor mRNA processing and hormone localization within cultured mouse fibroblast or rat pituitary cells.
Assuntos
Hormônio do Crescimento/genética , Animais , Sequência de Bases , Northern Blotting , Bovinos , Clonagem Molecular , Análise Mutacional de DNA , Éxons , Imunofluorescência , Expressão Gênica , Genes , Hormônio do Crescimento/metabolismo , Células L , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Processamento de Proteína Pós-Traducional , Splicing de RNA , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismoRESUMO
Human growth hormone (hGH) is a polypeptide with 191 amino acids and a molecular mass of 22 kilodaltons. With the aid of computer molecular simulation, an hGH analog was created by altering an hGH gene to reflect the change of one amino acid (glycine [G] 120 to arginine [R]) within the third alpha-helix of the hGH molecule. This hGH analog, named hGHG120R, was found to be an hGH antagonist. It may have important implications in treating human conditions in which hGH levels are abnormally high, as found in type I diabetics. Several hundred milligrams of purified hGHG120R were needed to determine the biological activity of the antagonist in animal models. A multistep downstream process was developed to purify hGHG120R from cultured mouse L cells transfected with the hGHG120R gene. The process consisted of cell clarification, salt precipitation, membrane ultrafiltration, reversed phase high performance liquid chromatography, phase separation, and lyophiliation. This work discusses the rationale for the design of the process and experimental results on the purification of hGHG120R using the process. (c) 1995 John Wiley & Sons, Inc.